Diagnostic agents for the prenatal diagnosis of preterm delivery, fetal infection, and fetal damage, and diagnostic kit containing the same

ABSTRACT

This invention is about a method for the prenatal diagnosis of preterm delivery, fetal infection, and fetal damage, and diagnostic reagent system and diagnostic kit for the diagnosis. The method, diagnostic reagent system, and kit are based on the finding that the level of MMP-8 in the amniotic fluid is significantly higher when the pregnant woman is at risk for preterm delivery, intrauterine infection, and fetal damage. The diagnostic reagent system and kit can be applied to patients with or without clinical signs of preterm labor or premature rupture of fetal membranes. With its superiority in sensitivity and specificity as well as its less invasiveness compared to the conventional method of measuring fetal blood cytokine levels, this diagnostic reagent system and kit is very useful in the prenatal diagnosis of preterm delivery, fetal infection, and fetal damage.

FIELD OF THE INVENTION

The present invention relates to a diagnostic reagent system for theprenatal diagnosis of preterm delivery, fetal infection, and fetaldamage, and a diagnostic kit using the same reagents and, moreparticularly, to the use of an amniotic fluid matrix metalloproteinase-8(MMP-8) concentration as a prenatal diagnostic marker for pretermdelivery, fetal infection, and fetal damage.

BACKGROUND OF THE INVENTION

It has long been recognized in the medical world that the prevention ofpreterm delivery or premature rupture of fetal membranes is preferred tothe post-treatment thereof. However, a great variety of factors are nowknown to cause preterm delivery or premature rupture of fetal membranes,making it difficult to prevent these undesirable events. The traditionalapproach to the prevention of preterm delivery has been identifying ahigh-risk group of women to which special attention should be paid basedon the knowledge of obstetrics and gynecology, demography, and varioussyndromes (Main et al., Am. J. Obstet. Gynecol., 151:892-898, 1985).However, this approach has the problem of being neither sensitive norspecific. To circumvent this problem, extensive research has beendirected to finding biochemical markers for the prediction of impendingpreterm delivery and premature rupture of fetal membranes, resulting inthe nomination of plasma estradiol-17 beta, progesterone, C-reactiveprotein as promising candidates. However, these candidates were found tobe of poor accuracy.

Besides the identification of such biochemical predictable markers,significant attention has been paid to the biochemical role of collagen,based on the fact that the chorionic membrane is composed of fibrousconnective tissue and the tensile strength of fibrous connective tissueis determined by its collagen content, as revealed through studies ofthe premature rupture of fetal membranes. On the basis of their findingsthat prematurely ruptured fetal membranes have low collagen contentcompared to normal membranes, some scientists concluded that thepremature rupture of fetal membranes is attributable to their lowtensile strength compared to that of normal fetal membranes (Obstet.Gynecol., 57:487-89, 1981). According to another study, it has beenreported that the serum activity of collagenase was high in prematurelyruptured fetal membranes and preterm labor (Obstet. Gynecol., 75:84-88,1990). However, the precise mechanism of such biochemical changes hasnot yet been elucidated (FEBBS Letters, 244(2):315-318, 1989).

Statistically, the frequency of preterm delivery before 37 weeks ofgestation is estimated to be about 8 to 10%. In Korea, about 50,000neonates are prematurely delivered every year. Preterm delivery oftencauses serious neonatal complications including sepsis, respiratorydistress syndrome, pneumonia, bronchopulmonary dysplasia,intraventricular hemorrhage, necrotizing enterocolitis and cerebralpalsy. The frequency and severity of such sequelae is greater theearlier the preterm delivery. Therefore, if preterm delivery isprevented, it will be possible to remarkably reduce the occurrence ofpremature neonates disabled by such diseases.

Recent reports disclose that at least 30 to 40% of preterm deliveriesare associated with intrauterine infection (Butler N R., Bonham D G.,Prenatal mortality. The first report of the British perinatal mortalitysurvey, Edinburgh, Churchill Livingstone, 115-145, 1963; Romero R.,Avila C., Sepulveda W., Preterm birth. Cause, prevention, andmanagement., McGrow-Hill Company, 97-136, 1993; Romero R., Mazor M.,Clin. Obstet. Gynecol., 31:553, 1990; Gibbs R S., Romero R., Hiller SL., et al., Am. J. Obstet. Gynecol., 166:1515, 1992).

Intrauterine infection may cause fetal damage by the following process.Intrauterine infection activates the maternal and fetal immune system tosecrete inflammatory mediators, such as cytokines from lymphocytes andMMPs (matrix-metalloproteinases) from neutrophils. When the inflammatorymediators reach a certain level, prostaglandin, which promotes uterinecontraction, is produced causing active labor leading to pretermdelivery. Additionally, increased levels of inflammatory mediators causethe fetus to be affected by fetal inflammatory response syndrome (FIRS).Inflammatory mediators cause sepsis or acute respiratory distresssyndrome or damage organs as a result of autoimmune diseases in adults.Likewise, fetal organs can be systemically injured by inflammatorymediators, resulting in brain white matter lesions and bronchopulmonarydysplasia. Therefore, the prenatal diagnosis of intrauterineinflammation is essential for the prevention of preterm delivery andfetal damage.

Generally, the prenatal diagnostic methods of intrauterine fetalinfection in current use are cordocentesis, in which fetal bloodcytokine levels are measured, and histologic examination of theumbilical cord to identify funisitis. However, cordocentesis is limitedin its usage due to its invasiveness, and funisitis can be diagnosedonly after delivery (Yoon B H., Romero R., Park J S., Kim C J., Choi JH., Han T R., Am. J. Obstet. Gynecol., 182:675-81, 2000; Yoon B H.,Romero R., Kim K S., Park J S., Ki S H., Kim B I., Jun J K., Am. J.Obstet. Gynecol., 181:773-9, 1999; Romero R., Gomez R., Ghezzi F., YoonB H., Mazor M., Edwin S S., Berry S M., Am. J. Obstet. Gynecol.,179:186-93, 1998).

The white blood cell count in the amniotic fluid is increased in casesof infection or inflammation of the amniotic cavity. Neutrophils in theamniotic fluid are considered to be of fetal origin (Knauper V., KramerS., Reinke H., Tschesche H., Eur. J. Biochem., 189:295-300, 1990; BlaserJ., Triebel S., Massjosthusmann U., Romisch J., Krahl-Mateblowski U.,Freudenberg W., Fricke R., Tschesche H., Clinic. Chim. Acta., 244:17-33,1996; Segura-Valdez L., Pardo A., Gaxiola M., Uhal B D., Becerril C.,Selman M., Chest., 117:684-94, 2000; Romanelli R., Mancini S.,Laschinger C., Overall C M., Sodex J., MaCulloch C A., Infect. Immun.,67:2319-26, 1999; Maymon E., Romero R., Pacora P., Gomez R., Athayde N.,Edwin S., Yoon B H., Am. J. Obstet. Gynecol., 183:94-9, 2000).Therefore, it is postulated that secretory products of neutrophils inamniotic fluid might reflect a fetal inflammatory response. The focus ofthe present invention is the level of MMP-8 in the amniotic fluid. Thedetermination of MMP-8 in amniotic fluid may be a marker of the fetalinflammatory response syndrome which can be diagnosed currently byhistologic examination of the umbilical cord after delivery or bycordocentesis with the determination of fetal blood cytokines.

MMP (matrix metalloproteinase) series, also collectively known asmatrixins, are zinc-dependent endopeptidases that function to degradeextracellular matrix proteins. These proteases constitute a large andgrowing family of proteins which share similar structures and enzymaticproperties. MMPs are broadly classified into five groups. Along withMMP-1 and MMP-13, MMP-8 belongs to an interstitial collagenase group.MMP-8 is similar in size to other interstitial collagenases but isglycosylated to a far greater extent. A fully glycosylated proenzymeform of MMP-8 has a molecular weight of 85 kDa. The proenzyme isconverted into an active form of 60-70 kDa with the loss of a 15-25 kDasegment. ProMMP-8 is activated in vitro by various proteinases,including trypsin, chymotrypsin and cathepsin G. Organomercurialcompounds were also found to activate proMMP-8. The activation mechanismof MMP-8 in vivo has not yet been fully clarified.

The prior technique concerning MMP-8 is found in U.S. Pat. No.5,736,341, which discloses methods and test kits capable of sensitiveand specific diagnosis of periodontal diseases based on monoclonalantibodies against MMP-8. The patent describes that since MMP-8 isdirectly associated with the destruction of periodontal connectivetissues during the progression of periodontitis and diffused into theoral cavity through the gingival pocket containing gingival crevicularfluid, measurement of the MMP-8 level in the oral cavity enables thesite-specific diagnosis of periodontitis. For the specific and sensitivebiochemical detection of periodontal diseases in progression, thesemethods measure the conversion of a proform of MMP-8 into an active formbecause the conversion takes place during the process of periodontalinfection. Nothing is mentioned about the use of MMP-8 in connectionwith preterm delivery and fetal infection and damage.

U.S. Pat. No. 5,641,636 refers to a method of predicting the onset offetal membrane rupture based on the activity of another matrixcollagenase, MMP-9, which belongs to a different group of enzymes fromthat of MMP-8. MMP-9 is a 92-kDa type IV collagenase/gelatinase orgelatinase B with the largest molecular weight among MMPs. Foractivation, the proenzyme form of MMP-9, i.e. proMMP-9, is initiallycleaved into an intermediate active form of about 83 kDa with concurrentproduction of a 9 kDa inactive cleavage fragment. The intermediateactive form is further processed proteolytically into an active form ofMMP-9 with 67 kDa (J. Biol. Chem., 267 (30):21712-21719, 1992). Theactivation of MMP-9 means the conversion into the 83 kDa intermediateactive form or the 67 kDa fully active form with gelatin degradationactivity. This patent measures the hydrolytic activity of MMP-9 indegrading denatured collagens, e.g. gelatins, to diagnose the prematurerupture of fetal membranes. However, because MMP-9 is already present inthe amniotic fluid before the onset of parturition, this method haslimited value in predicting the premature rupture of fetal membranes.

Approximately 30 to 40% of patients with preterm labor or prematurerupture of fetal membranes undergo preterm delivery. In this condition,various substances, including interleukin-6, interleukin-8, TNF-α,interleukin-1β, GROα, RANTES, white blood cells, MIP-1α, MCP-1, glucose,PGE₂, and angiogenin, are known to be present at increased levels in theamniotic fluid. However, these substances are of poor utility for theprediction of preterm delivery because their levels remain unchanged orare not detected in the amniotic fluid of pregnant women withoutclinical signs of preterm labor and are increased only after the onsetof preterm labor or premature fetal membrane rupture.

SUMMARY OF THE INVENTION

The intensive and thorough research on the prediction of pretermdelivery, fetal infection, and fetal damage was conducted by the presentinventors to overcome the problem of invasiveness of cordocentesis withthe determination of fetal blood cytokines and the problem ofpost-delivery identification of funisitis through histologic examinationof the umbilical cord. This resulted in the finding that the activity ofMMP-8 is detected in the amniotic fluid of women without clinical signsof preterm labor or preterm premature rupture of fetal membranes as wellas those with such signs, which allowed the development of a method andkit capable of diagnosing preterm delivery, fetal infection, and fetaldamage.

Therefore, it is the objective of the present invention to provide amethod and kit for the prenatal diagnosis of preterm delivery, fetalinfection, and fetal damage in pregnant women with or without clinicalsigns of preterm labor or premature rupture of fetal membranes, wherebyneonatal morbidity and serious complications or sequelae, such ascerebral palsy, can be prevented.

As one element of the present invention, a method is provided for theprenatal diagnosis of preterm delivery, fetal infection, and fetaldamage, by measuring the level of MMP-8 in the amniotic fluid inpregnant women with or without clinical signs of preterm labor orpremature rupture of fetal membranes.

As another element of the present invention, a diagnostic reagent systemand kit for the prenatal diagnosis of preterm delivery, fetal infection,and fetal damage is provided.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows distributions of amniotic fluid MMP-8 and IL-6concentrations in patients with spontaneous preterm delivery and withspontaneous full-term delivery.

FIG. 2 shows receiver operating characteristic curves in whichsensitivity versus specificity is plotted to select a cutoff value foramniotic fluid MMP-8 in the identification of spontaneous pretermdelivery.

FIG. 3 shows distributions of amniotic fluid MMP-8 concentrations inpatients with and without funisitis.

FIG. 4 shows a receiver operating characteristic curve in whichsensitivity versus specificity is plotted to select a cutoff value forMMP-8 in the identification of funisitis.

FIG. 5 shows distributions of amniotic fluid MMP-8 concentrations inpatients with and without prenatal development of cerebral palsy.

FIG. 6 shows a receiver operating characteristic curve in whichsensitivity versus specificity is plotted to select a cutoff value forMMP-8 in the identification of prenatal development of cerebral palsy.

DETAILED DESCRIPTION OF THE INVENTION

In one embodiment, the present invention pertains to determination ofthe activity of MMP-8 in the amniotic fluid, thereby prenatallydiagnosing preterm delivery, fetal infection, and fetal damage.

With activity to degrade extracellular matrix proteins, MMP-8 is azinc-dependent endopeptidase. Belonging to an interstitial collagenasegroup, MMP-8 can be purified from neutrophilic leukocytes in proenzymeform (proMMP-8). ProMMP-8 is highly glycosylated with a molecular weightof approximately 85 kDa. Activation of the proenzyme is achieved bycleavage of the proenzyme molecule, which creates the active collagenasewhose molecular weight varies from 60 to 70 kDa, depending on the modeof activation. The mechanism of activation in vivo likely involvesreactive oxygen species and oxidants such as hydroxyl radicals. When theN-terminal segment of the procollagenase are removed, the active site ofthe enzyme is generated and exposed.

MMP-8 is produced as a proenzyme form (proMMP-8) by neutrophilicleukocytes. ProMMP-8 exists in granules for storage and is secreted inresponse to stimuli. Secreted proMMP-8 is activated at extracellularinterstitium and degrades type I, II and III collagens with highspecificity for type I collagen. This enzyme, known as an importantmediator of inflammation-related tissue injury, is involved in thetissue injury caused by inflammatory diseases such as periodontitis,chronic obstructive pulmonary disease, rheumatoid arthritis, etc. Also,MMP-8 is known to be implicated in the progression of labor by causingthe effacement and dilatation of the uterine cervix.

Based on the finding that an elevated mid-trimester amniotic fluidconcentration of MMP-8 in women both with and without clinical signs ofpreterm labor or premature rupture of fetal membranes is highlyindicative of preterm delivery, the present inventors developed aprenatal diagnostic method of preterm delivery, fetal infection, andfetal damage.

In accordance with an embodiment of the present invention, a method forthe prenatal diagnosis of preterm delivery, fetal infection, and fetaldamage is provided, comprising the steps of:

1) sampling amniotic fluid from a pregnant woman; and

2) quantitatively measuring MMP-8 in the amniotic fluid sample.

Women with clinical signs of preterm labor or premature rupture of fetalmembranes as well as women without such clinical signs can be diagnosedfor preterm delivery.

The sampling of an amniotic fluid can be achieved by transabdominalamniocentesis under ultrasonographic guidance or other samplingprocesses. For quantitative determination of MMP-8 levels in theamniotic fluid, any analytic method, if based on antigen-antibodycoupling, can be used, and ELISA (enzyme-linked immunosorbent assay) ispreferred.

To determine if MMP-8 serves as a powerful clinical indicator for theprediction of preterm delivery in a mid-pregnancy stage, amniotic fluidconcentrations of MMP-8 were compared with those of IL-6, anotherexcellent indicator of inflammation, in patients who had a spontaneouspreterm delivery before 32 weeks of gestation or a term delivery aftermid-trimester amniocentesis without any clinical signs of impendingpreterm delivery. The amniotic fluid concentrations of MMP-8 and IL-6were significantly higher in those with preterm delivery. Patient withan amniotic fluid MMP-8 concentration higher than 23 ng/ml had aspontaneous delivery in 89% of cases, as seen in FIG. 1. In addition,statistical comparison demonstrated the superiority of MMP-8 to IL-6 insensitivity and specificity for the prediction of preterm delivery.Considering sensitivity, specificity and odds ratio as a whole, anamniotic fluid MMP-8 level of 23 ng/ml was selected as a cutoff suitablefor the prediction of preterm delivery.

An elevated mid-trimester amniotic fluid MMP-8 concentration higher thanthe cutoff means high risk of preterm delivery before 32 weeks ofgestation. Therefore, when genetic amniocentesis is conducted in amid-pregnancy stage, the quantification of amniotic fluid MMP-8 levelsis helpful in the identification of patients who are likely to undergo apreterm delivery.

To prove the usefulness of MMP-8 as a clinical predictor for fetalinfection and fetal damage, amniotic fluids taken from consecutivepatients who delivered preterm singleton neonates (gestational age <36weeks) within 72 hours of amniocentesis were cultured for aerobic andanaerobic bacteria and for mycoplasmas and amniotic fluid MMP-8concentrations were determined by ELISA. A histologic examination ofplacenta was made after delivery. MMP-8 concentrations were observed tobe significantly higher in patients with a positive amniotic fluidculture than in patients with a negative amniotic fluid culture. Also,the presence of histologic chorioamnionitis in placenta entailed asignificantly higher amniotic fluid MMP-8 level than the absence thereof(see data of Tables 1 to 4).

From these results, it can be inferred that the infection of amnioticfluid with microorganisms leads to such a significant increase inamniotic fluid MMP-8 level that quantification thereof can be diagnosticof intrauterine infection. As for histologic chorioamnionitis, it alsoinduces a significant increase of amniotic fluid MMP-8 level, so thatthe determination of the amniotic fluid MMP-8 level can reflect theintrauterine infection. In consequence, MMP-8 can be used as aneffective clinical predictor for the prenatal diagnosis of intrauterineinfection and inflammation as well as preterm delivery. Especially,quantification of amniotic fluid MMP-8 levels can diagnose intrauterineinflammation with higher specificity and positive predictive value.

In order to diagnose fetal infection straightforwardly, amniotic fluidconcentrations of MMP-8 were compared according to the presence orabsence of funisitis.

The fetal inflammatory response syndrome (FIRS) is a multi-systemdisorder associated with impending preterm delivery and adverse neonataloutcome. Regarded as the histologic counterpart of FIRS, inflammation ofthe umbilical cord (funisitis) has been associated with an increasedrisk for the development of cerebral palsy. Neutrophils found in theamniotic cavity are of fetal origin. Based on these findings, it ishypothesized that secretory products from neutrophils might be an indexof FIRS. To test this hypothesis, the relationship between amnioticfluid matrix metalloproteinase-8 (MMP-8) and funisitis was examined. TheMMP-8 concentration was observed to be significantly higher in thepresence of funisitis than in the absence of funisitis. The diagnosticindices of MMP-8 in the identification of funisitis were found to behigh in sensitivity, specificity, and negative predictive value (seeTables 5 and 6, and FIGS. 3 and 4).

From these results, it was found that there is a strong associationbetween amniotic fluid MMP-8 concentrations and fetal inflammation(funisitis). Therefore, the present invention proposes that assessmentof amniotic fluid MMP-8 concentrations may assist the diagnosis of thefetal infection without resorting to conventional invasive fetal bloodsampling.

With the evidence of the correlation between the amniotic fluid MMP-8concentration and fetal infection, the present inventors studied therelationship between amniotic fluid MMP-8 concentrations and neonatalmorbidity, which includes neonatal sepsis, respiratory distresssyndrome, pneumonia, bronchopulmonary dysplasia, necrotizingenterocolitis, and intraventricular hemorrhage. A significantly higherMMP-8 concentration was seen in the presence of neonatal morbidity thanin the absence of neonatal morbidity (see Table 7). The diagnosticindices of MMP-8 in the identification of neonatal morbidity wereexcellent in specificity, and positive and negative predictive values(see Table 7 and 8). Additionally, the amniotic fluid MMP-8concentration was significantly higher in patients who deliveredneonates with neonatal sepsis than in those who delivered neonateswithout neonatal sepsis (P<0.05) (see Table 9).

The data, taken together, demonstrates that amniotic fluid MMP-8concentration can be useful as a diagnostic marker in the prediction ofneonatal sepsis and perinatal morbidity.

Intrauterine infection or inflammation has been implicated in theetiology of cerebral palsy. FIRS is believed to cause fetal brain damagein term and preterm birth. Neutrophils are the cells most frequentlyrecruited into the amniotic fluid in cases of infection and they areconsidered to be of fetal origin. The amniotic fluid level of MMP-8, anenzyme secreted by activated neutrophils, was found to be significantlyhigher in cases of intrauterine infection and/or inflammation, asdemonstrated in the following Examples 2 and 3. Based on thisbackground, an examination was made to determine if increasedconcentrations of matrix metalloproteinase-8 (MMP-8) in amniotic fluidare associated with the development of cerebral palsy at the age ofthree.

A significantly higher level of MMP-8 was observed in the amniotic fluidtaken from the patients whose newborns developed cerebral palsy,compared to those whose newborns did not develop cerebral palsy (seeFIG. 5), demonstrating that MMP-8 is very useful as a diagnostic markerfor the prediction of cerebral palsy (see FIGS. 5 and 6, and Table 11).

We could see that the elevated amniotic fluid MMP-8 concentration isassociated with odds that are six-fold higher of developing cerebralpalsy. That is, the amniotic fluid MMP-8 concentration can be used as aprenatal diagnostic marker for the prediction of cerebral palsy.

After extensive studies, a cutoff value of amniotic fluid MMP-8 in theidentification of preterm delivery, fetal infection, and fetal damagewas selected. In this regard, the pregnant woman is identified as beingat risk for preterm delivery, neonatal morbidity, fetal infection(funisitis), and cerebral palsy when the cutoff value of amniotic fluidMMP-8 is higher than 5-100 ng/ml and preferably higher than 10-50 ng/ml.

Based on the usefulness of the amniotic fluid MMP-8 concentration as adiagnostic marker in the identification of preterm delivery, fetalinfection, and fetal damage, a reagent system can be developed fordiagnosing preterm delivery, fetal infection, and fetal damage.

Therefore, in another embodiment of the present invention, a diagnosticreagent system for the identification of preterm delivery, fetalinfection, and fetal damage, which is based on the quantification ofamniotic fluid MMP-8 concentrations, is provided. In detail, thediagnostic reagent system makes use of an analytic mechanism comprisingthe steps of:

1) adsorbing primary MMP-8 antibodies onto a matrix; and

2) incubating the MMP-8 antibodies adsorbed onto the matrix in thepresence of an amniotic fluid and washing the matrix to remove unboundantigens;

3) coupling a secondary chromogenic enzyme- or fluorescent-linkedantibody to the MMP-8 bound to the primary antibodies adsorbed onto thematrix; and

4) developing a chromogenic reaction in the matrix by use of a coloringagent with quantitative analysis of the amount of antibody-bound MMP-8.

Examples of the matrix useful in step 1 include a nitrocellulosemembrane, a 96-well plate of polyvinyl resin, a 96-well plate ofpolystyrene resin, and a glass slide.

For quantitative analysis, the secondary antibody to be coupled to theMMP-8 bound to the primary antibody is linked to a chromogenic enzyme,such as peroxidase, alkaline phosphatase, and biotin, or a fluorescentagent such as FITC (Fluorescein Isothiocyanate) and TRITC.

The coloring agent can be selected from a group of agents consisting of4CN (4-chloro-1-naphthol), DAB (diaminobenzidine), AEC (aminoethylcarbazol), ABTS (2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonicacid)), OPD (O-phenylenediamine) and TMB (tetramethyl benzidine).

In principle, the diagnosis is based on the quantification of amnioticfluid MMP-8 concentrations, which takes advantage of the reaction ofMMP-8 to its antibodies. In this connection, monoclonal or polyclonalanti-MMP-8 antibodies are immobilized to a solid matrix and reacted toMMP-8 in a sample, followed by washing the matrix to remove unboundantibodies and MMP-8. Then, a secondary monoclonal or polyclonalantibody which is linked with an enzyme or a fluorescent is bound to theimmobilized MMP-8. A horseradish peroxidase polyclonal antibody or abiotinylated rabbit polyclonal or monoclonal antibody is usually used asthe secondary antibody. A chromogenic reaction for visualization isdeveloped in the presence of a peroxide and a coloring agent. Additionof an acid halts the chromogenic reaction, followed by the measurementof absorbance at 450 nm.

Based on this diagnostic mechanism, a diagnostic kit can be configured,which uses the same diagnostic reagent system.

Therefore, in a further embodiment of the present invention, adiagnostic kit for the identification and prediction of pretermdelivery, fetal infection, and fetal damage is provided. With the aid ofthe diagnostic kit, preterm labor or premature rupture of fetalmembranes can be assessed quantitatively or qualitatively withconvenience. For the assay of an MMP-8 antigen, a marker antibody isprepared by coupling gold or colloid particles to an anti-MMP-8antibody. The marker antibody against MMP-8 is bound to MMP-8 to form animmune complex. This immune complex is again reacted with the MMP-8antibody, and then washing fluid for excess marker antibody, usuallymade of urea etc., is added. A positive result can be visualized by theeye when the MMP-8 concentration exceeds a certain level. Such adiagnostic kit may comprise anti-MMP-8 antibody, standard MMP-8, matrix,assay buffer, chromogenic enzyme- or fluorescent-labeled secondaryantibody, and adhesive plate cover.

Alternatively, the diagnostic kit of the present invention may adopt anautomated analytic method using a biological microchip. For instance, adiagnostic kit can be structured to perform immunoblotting using ananti-MMP-8 antibody-coated slide glass. This diagnostic kit may comprisea biological microchip with an anti-MMP-8 antibody immobilized onto itssurface, appropriate buffer, standard MMP-8, and secondary antibody.

EXAMPLES

A better understanding of the present invention may be obtained in lightof the following examples which are set forth to illustrate, but are notto be construed to limit the present invention.

Example 1 Selection of Diagnostic Cutoff Value for Prenatal Diagnosis ofPreterm Delivery

Strong evidence implicates chronic intrauterine infection in theetiology of preterm delivery. In this example, an examination wasconducted to determine if amniotic fluid concentrations of MMP-8 andinterleukin-6 (IL-6) can be used to identify patients at risk forspontaneous preterm delivery among pregnant women without clinical signsof preterm delivery.

To this end, a case-control study was designed with stored amnioticfluid obtained from women who had mid-trimester genetic amniocentesis.Amniotic fluid levels of MMP-8 and IL-6 were determined by ELISA in 19patients with a spontaneous preterm delivery before 32 weeks ofgestation, and 95 patients who delivered normal neonates at full-term ascontrol cases. Cases with an abnormal fetal karyotype and majoranomalies were excluded from this analysis.

The median amniotic fluid MMP-8 concentration with spontaneous pretermdelivery was 3.1 [0.3-1954.9] ng/ml, while the level was 1.3 [<0.3-45.2]ng/ml in the control cases. The median amniotic fluid IL-6 concentrationwas 0.32 [0.04-2.52] ng/ml in patients with spontaneous preterm deliverywhile the level was 0.18 [0.01-1.81] ng/ml in the control cases. BothMMP-8 and IL-6 concentrations of amniotic fluid taken at mid-pregnancystage were significantly higher in patients with spontaneous pretermdelivery than in the control cases with full-term delivery (p<0.01), asshown in FIG. 1.

After the assessment of MMP-8 concentrations in patients and the controlcases, when the amniotic fluid MMP-8 concentration at a mid-pregnancystage was above 23 ng/ml, 89% of the cases investigated had aspontaneous preterm delivery.

Additionally, an amniotic fluid MMP-8 cutoff value of 23 ng/ml at amid-pregnancy stage showed a sensitivity of 42% (8/19) and a specificityof 99% (94/95) in the identification of the patients with early pretermdelivery after genetic amniocentesis, while an IL-6 cutoff value of 0.6ng/ml showed a sensitivity of 42% (8/19) and a specificity of 92%(87/95), as shown in FIG. 2. MMP-8 was therefore superior to IL-6 insensitivity, specificity and odds ratio for the identification andprediction of preterm delivery. Consequently, an amniotic fluid MMP-8concentration of 23 ng/ml was selected as a cutoff value in theidentification of prematurity.

Example 2 Relationship Between Amniotic Fluid Concentration of MMP-8 andIntrauterine Infection and Inflammation

Amniotic fluid concentrations of MMP-8 were examined in the presence orabsence of intrauterine infection and inflammation to determine if theamniotic fluid MMP-8 concentration could be used as a diagnostic markerin the identification of intrauterine infection and fetal damage.

In this regard, 255 consecutive patients who delivered preterm singletonneonates (gestational age <36 weeks) within 72 hours of amniocentesis atSeoul National University Hospital in Seoul, Korea were examined. Theamniotic fluids were measured for their MMP-8 levels as well as culturedfor aerobic and anaerobic bacteria and mycoplasmas. In addition,histologic examination of the placenta was performed. Amniotic fluid wasretrieved by transabdominal amniocentesis under ultrasonographicguidance.

2-1: Amniotic Fluid Culture

Immediately after being retrieved by transabdominal amniocentesis, theamniotic fluid was put into sterile plastic vessels with caps and storedtherein until culturing in anaerobic or aerobic media. Useful inculturing aerobes or anaerobes were blood agar, McConkey's agar, Bactec6A vial, thioglycollate broth, brucellar blood agar, fresh meatextracts, and pleuropneumoni-like organism broth supplemented with horseserum penicillin polymixin B and amphotericin B. Mycotrim GU was used toculture mycoplasmas.

2-2: Measurement of Amniotic Fluid MMP-8 Concentration

Amniotic fluid samples were centrifuged at 700×g for 10 min. Thesupernatant was used to measure the amniotic fluid MMP-8 concentrationwith ELISA (Amersham Pharmacia Biotech, UK) using two monoclonalantibodies which bind to non-overlapping epitopes.

2-3: Histologic Examination of Placenta

After the placenta was fully drawn out at delivery, tissues excised fromthe umbilical cord, the chorionic plate, and the placental membrane werefixed in 10% formalin and embedded in paraffin to prepare slides.Afterwards, the tissue segments were dyed with hematoxylin and eosin forvisualization under a microscope. Acute intrauterine inflammation wasdefined as the presence of inflammatory changes on examination of any ofthe fetal membrane, chorionic membrane, decidua and the chorionic plate.

Of the 255 subjects, 45 cases were found to have positive amniotic fluidculture, which shows the intrauterine infection frequency to be 18%. Onhistologic examination of the placenta 113 patients were found to havechorioamnionitis, which indicates the intrauterine inflammationfrequency to be 44%. The median amniotic fluid MMP-8 concentration wasdetected at a level of 191.4 ng/ml in patients with a positive amnioticfluid culture of bacteria and at a level of 2.7 ng/ml in those with anegative culture. Therefore, the median amniotic fluid MMP-8concentration in patients with intrauterine infection was significantlyhigher than those without intrauterine infection (p<0.001). The resultsare given in Table 1, below.

TABLE 1 Amniotic Fluid MMP-8 Concentration According to Amniotic FluidCulture Result Bacteria in Amniotic Fluid Median Value (ng/mL) Interval(ng/mL) Positive 191.4 <0.3–4202.7 Negative 2.7 <0.3–3929.0

With a cutoff value of 23 ng/ml, the diagnostic indices of MMP-8 for theidentification of positive amniotic fluid cultures were excellent:sensitivity of 76% and negative predictive value of 93%. The results aregiven in Table 2, below.

TABLE 2 Diagnostic Indices of MMP-8 for the Identification of PositiveAmniotic Fluid Culture Sensitivity 76% Specificity 70% PositivePredictive Value 35% Negative Predictive Value 93%

Histologic examination of the placenta revealed that the medianconcentration of amniotic fluid MMP-8 was 160.9 ng/ml in the presence ofchorioamnionitis, but 1.0 ng/ml in the absence of chorioamnionitis. Theamniotic fluid MMP-8 concentration was significantly higher in patientswith chorioamnionitis than those without chorioamnionitis (p<0.001). Theresults are given in Table 3, below.

TABLE 3 MMP-8 Concentration According to Intrauterine InflammationHistologic Chorioamnionitis Median Value (ng/mL) Interval (ng/mL)Present 160.9 <0.3–4202.7 Absent 1.0 <0.3–766.2

With a cutoff value of 23 ng/ml, the diagnostic indices of MMP-8 for theidentification of intrauterine inflammation (chorioamnionitis) wereexcellent: sensitivity of 72%, specificity of 89%, positive predictivevalue of 84%, and negative predictive value of 80%. The results aregiven in Table 4, below.

TABLE 4 Diagnostic Indices for the Identification of IntrauterineInflammation Sensitivity 72% Specificity 89% Positive Predictive Value84% Negative Predictive Value 80%

Example 3 Diagnosis of Funisitis by Use of Amniotic Fluid Concentrationof MMP-8

Amniotic fluid MMP-8 concentrations were measured in the presence or inthe absence of funisitis to determine if amniotic fluid MMP-8concentrations could be utilized to directly diagnose fetal infection.

The relationship between the presence of funisitis and amniotic fluidconcentrations of MMP-8 was examined in 255 consecutive patients whodelivered preterm singleton neonates (gestational age <36 weeks) within72 hours of amniocentesis at the Seoul National University Hospital inSeoul, Korea. Funisitis was diagnosed by the presence of neutrophilinfiltration into the umbilical vessel walls or Wharton jelly.Quantification of MMP-8 concentration was conducted in the same manneras in Example 2.

Funisitis was diagnosed in 59 cases (funisitis frequency 23%). Themedian amniotic fluid MMP-8 concentration was 433.7 ng/ml in patientswith funisitis and 1.9 ng/ml in those without funisitis. Therefore, thepatients with funisitis had a significantly higher median amniotic fluidMMP-8 concentration compared to those without funisitis (p<0.001). Theresults are given in Table 5, below and FIG. 3.

TABLE 5 MMP-8 Concentration According to Presence or Absence ofFunisitis Funisitis Median Value (ng/mL) Interval (ng/mL) Present 433.7  1.5–3836.8 Absent 1.9 <0.3–4202.7

Receiver operating characteristic curve analysis was employed to selecta cutoff value for amniotic fluid analytes in the diagnosis offunisitis. As a result, a cutoff of 23 ng/ml was selected for MMP-8 inconsideration of both sensitivity and positive predictive value for thediagnosis of funisitis, as seen in FIG. 4. The diagnostic indices ofMMP-8 (cutoff 23 ng/ml) in the identification of funisitis wereexcellent: sensitivity of 90%, specificity of 78%, and negativepredictive value of 96%. The results are summarized in Table 6, below.

TABLE 6 Diagnostic Indices of MMP-8 for the Identification of FunisitisSensitivity 90% (53/59) Specificity 78% (153/196) Positive PredictiveValue 55% (53/96) Negative Predictive Value 96% (153/159)

Example 4 Diagnosis of Neonatal Morbidity by Use of Amniotic Fluid MMP-8Concentration

Based on the postulation that funisitis is associated with an increasedrisk of neonatal infection-related complications such as sepsis,pneumonia, bronchopulmonary dysplasia, necrotizing enterocolitis, andintraventricular hemorrhage, amniotic fluid MMP-8 concentrations incases with such neonatal morbidity were compared with those in normalcases.

The relationship between the presence of such neonatal morbidity andamniotic fluid concentration of MMP-8 was examined in 239 consecutivepatients who delivered preterm singleton neonates (gestational age <36weeks) within 72 hours of amniocentesis at the Seoul National UniversityHospital in Seoul, Korea. Neonatal morbidity was defined by thedevelopment of neonatal complications, such as sepsis, pneumonia,bronchopulmonary dysplasia, necrotizing enterocolitis, andintraventricular hemorrhage. Congenital neonatal sepsis was diagnosed inthe presence of a positive blood culture within 72 hours of delivery.The diagnosis of neonatal respiratory distress syndrome required thepresence of respiratory grunting and retracting, an increased oxygenrequirement (FiO₂>0.4), and diagnostic radiographic and laboratoryfindings without the evidence of other causes of respiratory disease.Pneumonia was diagnosed in the presence of definite clinical andradiologic findings, with or without a positive culture from trachealaspirate or chest tube specimen within 7 days of birth. Bronchopulmonarydysplasia was diagnosed by the criteria proposed by Bancalari et al.:(1) Intermittent positive-pressure ventilation was required during thefirst week of life and for a minimum of 3 days; (2) clinical signs ofchronic respiratory disease developed, characterized by tachypnea,intercostal and subcostal retraction, and rales on auscultation, allpersisting for longer than 28 days; (3) supplemental oxygen was requiredfor more than 28 days to maintain a PaO₂ over 50 mmHg; (4) chestradiograph showed persistent strands of densities in both lungs,alternating with areas of normal or increased lucency. In some cases, anautopsy was needed for the diagnosis of bronchopulmonary dysplasia.Intraventricular hemorrhage was graded according to the system proposedby McMenamin et al. Necrotizing enterocolitis was diagnosed in thepresence of abdominal expansion and feeding intolerance for at least 24hours with radiologic findings of air entrapment in enteric walls,intestinal rupture, and meconium obstructive syndrome, or by operationalor autopsic findings of necrotic intestinal rupture. Quantification ofamniotic fluid MMP-8 concentrations was conducted in the same manner asin Example 2.

107 neonates of the 239 subjects (frequency 45%) were diagnosed assuffering from neonatal morbidity. The median amniotic fluid MMP-8concentration remained at a level of 2.35 ng/ml in the absence of majorneonatal morbidity while a significantly higher MMP-8 concentration(160.9 ng/ml) was observed in the presence of major neonatal morbidity(p<0.001). Details are given in Table 7, below.

TABLE 7 MMP-8 Concentration According to the Presence or Absence ofNeonatal Morbidity Neonatal Morbidity Median Value (ng/mL) Interval(ng/mL) Present 17.0 <0.3–4202.7 Absent 2.35 <0.3–1333.1

Receiver operating characteristic curve analysis was employed to selecta cutoff value for amniotic fluid analytes in the diagnosis of neonatalmorbidity. As a result, a cutoff of 23 ng/ml was selected for MMP-8considering sensitivity and positive predictive value for the diagnosisof neonatal morbidity. The diagnostic indices of MMP-8 (cutoff 23 ng/ml)for the identification of neonatal morbidity were excellent: specificityof 77%, positive predictive value of 63% and negative predictive valueof 65%. The results are summarized in Table 8, below.

TABLE 8 Diagnostic Indices of MMP-8 for the Identification of NeonatalMorbidity Sensitivity 50% Specificity 77% Positive Predictive Value 63%Negative Predictive Value 65%

Additionally, the median amniotic fluid MMP-8 concentration wassignificantly higher in the presence of sepsis (208.95 ng/ml), comparedto the absence of sepsis (4.4 ng/ml) (p<0.05). Details are given inTable 9, below.

TABLE 9 MMP-8 Concentration According to the Presence or Absence ofNeonatal Sepsis Neonatal Sepsis Median Value (ng/mL) Interval (ng/mL)Present 208.95   2.4–1568.6 Absent 4.4 <0.3–4202.7

The diagnostic indices of MMP-8 (cutoff 23 ng/ml) in the identificationof neonatal sepsis were excellent: sensitivity of 67%, specificity of66%, and negative predictive value of 99%. The results are summarized inTable 10, below.

TABLE 10 Diagnostic Indices of MMP-8 for the Identification of NeonatalSepsis Sensitivity 67% Specificity 66% Positive Predictive Value  5%Negative Predictive Value 99%

Example 5 Diagnosis of Cerebral Palsy by Use of Amniotic Fluid MMP-8Concentration

The relationship between amniotic fluid concentrations of MMP-8 and thedevelopment of cerebral palsy was examined in 116 preterm singletonnewborns (gestational age at birth, <35 weeks) born to mothers whounderwent amniocentesis and were followed for at least three years.Cerebral palsy was diagnosed in the presence of a definite abnormalityon the neurodevelopmental assessment (abnormality of developmentalmilestone, postural abnormality by the Vojta method, and reflexabnormality) and a persistent abnormality of muscle tone.

Median amniotic fluid concentration of MMP-8 was significantly higher inmothers whose newborns developed cerebral palsy than in mothers whosenewborns did not develop cerebral palsy (median 153.9 [range<0.3-1535.9] ng/ml versus median 6.4 [range <0.3-3836.8] ng/ml; p<0.01).Neonates who developed cerebral palsy were delivered at earliergestational age than those without cerebral palsy. After adjustment forthe gestational age at birth and the results of amniotic fluid culture,elevated concentrations of amniotic fluid MMP-8 significantly increasedthe odds of development of cerebral palsy (odds ratio, 6.0; 95%confidence interval, 1.1-33.0; p<0.05).

Receiver operating characteristic curve analysis was employed to selecta cutoff value for amniotic fluid analytes in the diagnosis of cerebralpalsy. As a result, a cutoff of 23 ng/ml was selected for MMP-8 inconsideration of both sensitivity and positive predictive value for thediagnosis of cerebral palsy, as shown in FIG. 6. The diagnostic indicesof MMP-8 (cutoff 23 ng/ml) in the diagnosis of cerebral palsy wereexcellent: sensitivity of 85%, specificity of 69%, and negativepredictive value of 97%. The results are summarized in Table 11, below.

TABLE 11 Diagnostic Indices of MMP-8 for Cerebral Palsy Sensitivity 85%Specificity 69% Positive Predictive Value 26% Negative Predictive Value97%

INDUSTRIAL APPLICABILITY

As described hereinbefore, a diagnostic reagent system and a diagnostickit are provided for the prenatal diagnosis of preterm delivery, fetalinfection, and fetal damage, based on the quantification of amnioticfluid MMP-8 concentration. Taking advantage of an antigen-antibodyreaction coupled with chromogenesis, the diagnostic reagent system anddiagnostic kit of the present invention is characterized by comprisingone or more anti-MMP-8 antibody. The diagnostic reagent system and kitof the present invention can be applied to patients with or withoutclinical signs of preterm delivery or premature rupture of fetalmembranes. Superior in sensitivity and specificity to conventionalmethods of measuring fetal blood cytokine levels, the present inventionis very useful in prenatal diagnosis of preterm delivery, fetalinfection, and fetal damage.

1. A diagnostic method for prenatal identification of an increased riskof preterm delivery of a pregnant woman without clinical symptoms ofpreterm labor or preterm premature rupture of membrane at midtrimester,comprising the steps of: 1) sampling amniotic fluid from the pregnantwoman at midtrimester; and 2) measuring matrix metalloproteinase-8 levelin the amniotic fluid sample by an immunoassay, and determining that thematrix metalloproteinase-8 level is greater than about 23 ng/ml in theamniotic fluid as an indication that the pregnant woman is at increasedrisk for preterm delivery.
 2. The method according to claim 1, whereinstep 2) comprises the steps of: 1) adsorbing primary anti-matrixmetalloproteinase-8 antibodies onto a matrix, 2) incubating the primaryanti-matrix metalloproteinase-8 antibodies adsorbed onto the matrix withthe amniotic fluid sample, 3) coupling chromogenic enzyme- orfluorescent agent-linked secondary anti-matrix metalloproteinase-8antibodies to the matrix metalloproteinase-8 that is bound to theprimary antibodies adsorbed onto the matrix, 4) developing a chromogenicreaction with a coloring agent and measuring absorbance of thechromogenic reaction when the chromogenic enzyme-linked antibodies areused, or measuring fluorescence when the fluorescent agent-linkedantibodies are used in the matrix as an indication of the matrixmetalloproteinase-8 level in the amniotic fluid sample; and 5)determining whether matrix metalloproteinase-8 level is greater thanabout 23 ng/ml.
 3. The method according to claim 2, wherein theanti-matrix metalloproteinase-8 primary and secondary antibodies areindependently monoclonal or polyclonal.
 4. The method according to claim3, wherein the primary and secondary antibodies are monoclonal.
 5. Themethod according to claim 2, wherein the matrix is selected from thegroup consisting of a nitrocellulose membrane, a 96-well plate ofpolyvinyl resin, a 96-well plate of polystyrene resin, and a glassslide.
 6. The method according to claim 2, wherein the chromogenicenzyme is selected from the group consisting of peroxidase and alkalinephosphatase.
 7. The method according to claim 2, wherein the fluorescentagent is selected from the group consisting of FITC and TRITC.
 8. Themethod according to claim 2, wherein the coloring agent is selected fromthe group consisting of 4-chloro-1-naphtol, diaminobenzidine, aminoethylcarbazole, 2,2′-azion-bis(3-ethylbenzothiazoline-6-sulfonic acid),o-phenylenediamine, and tetramethyl benzidine.